In type 1 diabetes autoreactive T cells mediate the destruction of insulin producing beta cells. A key question in the study of this disease is why T cells, which are there to protect us from pathogens such as viruses, turn against the body?s own cells. Protein modifications that form within cells provide a plausible explanation for this misguided attack as the composition of proteins is altered, making them appear foreign to the immune system. Using mass spectrometric analyses on beta cell extracts, we recently identified a new family of protein modifications that forms in beta cells and is targeted by disease driving T cells in a major animal model of type 1 diabetes. Furthermore, we demonstrated that autoreactive T cells, that were isolated from the residual islets of type 1 diabetic organ donors, also target proteins carrying this type of modification. The modification is a result of insulin fragments that are cross-linked to other protein fragments, leading to the formation of hybrid peptides. At the junction those peptides contain new amino acid sequences that are not encoded by the organism?s genome. These non-germline encoded sequences deliver plausible targets for autoreactive T cells that mediate the destruction of beta cells. Here we will (1) search for hybrid peptides in human beta cells by mass spectrometry, (2) study the mechanism that leads to the formation of hybrid peptides, and (3) devise new bioinformatics tools that will allow us to confidently and rapidly identify hybrid peptides in beta cells by searching mass spectrometric datasets. Identification of hybrid peptides in human tissue will provide us with valuable tools to devise new strategies to predict, prevent or reverse type 1 diabetes. Understanding the mechanism that leads to the formation of hybrid peptides will allow us to test strategies which prevent the formation of those peptides, rendering beta cells invisible to disease driving T cells. Bioinformatics tools will allow us to characterize the vast number of potential hybrid peptides that may form in beta cells as well as additional cells types that are targeted in other autoimmune diseases. In summary, success in any of the aims of this grant will provide us with valuable tools and reagents to study type 1 diabetes in unprecedented detail.